The applicability of human monoclonal antibodies (HuMAbs), especially HuMAbs to human cytokines, in therapy holds great promise; see, for example, Griffiths et al., EMBO J., 12:725-734 (1993) and the review in Larrick et al., J. Biol. Response Modif., 5:379 (1986). However, the production of useful HuMAbs against human cytokines has proved difficult.
Specifically, while the possible existence in human serum of autoantibodies to human cytokines is mentioned in numerous articles Suzuki et al., J. Immunol., 145:2140-2146 (1990) (IL-1.alpha.); Hansen et al., Immunol. Letters, 30:133-140 (1991) (IL-1.alpha.); Bendtzen et al., Immunol. Today, 11:167-169 (1990) (IL-1.alpha. and TNF-.alpha.); Bendtzen et al., Immunol. Today, 10:222 (1989) (IL-1.alpha. and TNF-.alpha.); Saurat et al., J. Allergy Clin. Immunol., 88:244-256 (1991) (IL-1.alpha.); Suzuki et al., Clin. Exp. Immunol., 85:407-412 (1991) (IL-1.alpha.); Sunder-Plassmann et al., Kidney International, 40:787-791 (1991) (IL-1.alpha.); Gallay et al., Eur. Cytokine Netw., 2:329-338 (1991) (IL-1.alpha. and IL-1.beta.); Mae et al., Lymphokine Cytokine Res., 10:61-68 (1991) (IL-1.alpha.); Fomsgaard et al., Scand. J. Immunol., 30:219 (1989) (TNF-.alpha.); Hansen et al., Scand. J. Immunol., 33: 777-781 (1991) (IL-6); Crabtree et al., Scand. J. Immunol., 37:65-70 (1993) (IL-8); Bost et al., Immunology, 65:611-615 (1988) (IL-2); Ross et al., Clin. Exp. Immunol., 82:57-62 (1990) (IFN-.alpha.2b and IFN-.gamma.); and Caruso et al., J. Immunol., 144:685-690 (1990) (IFN-.gamma.)!, no one has been able to produce an isolated and purified HuMAb to a human cytokine, especially a HuMAb having high affinity, e.g., a K.sub.a of above about 10.sup.9 M.sup.-1. Some of the reasons are pointed out in the cited article by Griffiths et al. in EMBO. J.:
"Human monoclonal antibodies (mAbs) have huge potential for therapy, but are difficult to make by immortalizing B-lymphocytes. Furthermore, it is especially difficult to generate human mAbs directed against human antigens (anti-self antibodies), for example antibodies against soluble TNF to block septic shock, against membrane-bound carcinoembryonic antigen to image colorectal carcinoma, or against lymphocyte antigens to destroy tumour in lymphoma. This difficulty results from immunological tolerance mechanisms that prevent the antigen-driven expansion of B-cell clones with self specificities. After antibody gene rearrangement, virgin B-cells may display antibodies with self-reactivity, but tolerance mechanisms can lead to their deletion or to their anergy. It has been suggested that cells may be anergized if the antigen is soluble, but deleted if the antigen is membrane bound. B-cell tolerance does not seem to occur when concentrations of soluble antigen are low (in contrast to T-cell tolerance) and B-cells with poor affinities for antigen are not tolerized, even at higher antigen concentrations. Such non-tolerized B-cells are not usually expanded because they lack T-cell help, although proliferation can be induced artificially by using polyclonal B-cell activators. PA1 It is estimated that 10-30% of B-lymphocytes in normal, healthy individuals are engaged in making autoantibodies. However, the `natural autoantibodies` produced do not lend themselves to therapeutic use as they are often IgM, low affinity and polyreactive." (Citations omitted.) PA1 a V.sub.H segment having an amino acid sequence defined by amino acids 1-122 of an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO. 1 or by a CDR somatic variant thereof, and/or PA1 a V.sub.L segment having an amino acid sequence defined by amino acids 1-108 of an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO. 2 or by a CDR somatic variant thereof. PA1 a nucleotide sequence defined by base numbers 58-423 of SEQ ID NO. 1 or by a CDR encoding somatic variant thereof, or a functional equivalent of such a nucleotide sequence, and/or PA1 a nucleotide sequence defined by base numbers 67-390 of SEQ ID NO. 2 or by a CDR encoding somatic variant thereof; or PA1 a functional equivalent of one or both of said nucleotide sequences. PA1 (1) contacting the solution with labeled protein and polyclonal or monoclonal anti-human Ig (i.e., anti-IgA, IgD, IgE, IgG and/or IgM) coupled to a substrate or with labeled protein and protein G coupled to a substrate; and PA1 (2) determining if a desired human monoclonal antibody is present in the solution by detecting labeled protein in any immunoprecipitated product. PA1 serially diluting a human B cell mixture giving a positive result in the screen to provide a purified mixture of human B cells or single B cell clones; culturing said purified mixture of human B cells or single human B cell clones; and PA1 screening supernatants from said cultured purified mixture of human B cells or single B cell clones by the above methods to determine if the desired human monoclonal antibody is present in the supernatants of said cultured purified mixture of human B cells or single B cell clones. PA1 producing a CD40-crosslinked and EBV-transformed, immortalized and/or activated B cell population containing immortalized and/or activated B cells expressing said human monoclonal antibody; PA1 cloning subpopulations of said immortalized and/or activated B cell population and identifying a subpopulation which contains immortalized and/or activated B cells expressing said human monoclonal antibody; PA1 preparing a cDNA library using the mRNA from said subpopulation to create a repertoire of DNAs encoding at least the V.sub.H and/or V.sub.L chain of the human monoclonal antibodies expressed by said subpopulation of immortalized and/or activated B cells. PA1 identifying DNA within said library that encodes at least the V.sub.H and/or V.sub.L chain of the desired human monoclonal antibody; and PA1 using said DNA to produce a human monoclonal antibody against the desired antigen or an antigen-binding fragment of such an antibody. PA1 establishing an immortalized and/or activated human B cell population from a patient having antibodies that bind to the human cytokine, said immortalization and/or activation comprising infecting the B cells with Epstein-Barr virus and crosslinking the CD40 of such B cells; PA1 culturing said immortalized and/or activated B cells; PA1 isolating multiple clones from such immortalized and/or activated B cells, each of which clones secretes a human monoclonal antibody that binds to the cytokine; and PA1 using one or more of such clones to produce one or more human monoclonal antibody or a fragment thereof.
Although the Griffiths et al. article speaks of "human self-antibodies with high specificity," only single-chain V.sub.H and V.sub.L fragments are actually disclosed. Moreover, there is no disclosure in the article that any of the heavy/light-chain combinations mentioned therein are actually from one human antibody. Moreover, the human antibody fragments disclosed all have relatively low affinities, i.e., K.sub.a s below 2.times.10.sup.7 M.sup.-1 and most below 10.sup.7 M-.sup.1.